HC3: Cancer biology

Hallmarks

There are 10 hallmarks of cancer which distinguish epithelial cells from carcinomas:

1. Deregulating cellular energetics
2. Sustaining proliferative signaling
3. Evading growth suppressors
4. Avoiding immune destruction
5. Enabling replicative immortality
6. Activating invasion and metastasis
7. Inducing angiogenesis
8. Resisting cell death
9. Genome instability and mutation
10. Tumor promoting inflammation

Tumor promoting inflammation and genome instability and mutation are enabling characteristics → enhance the cancer development progress. The other hallmarks are fundamental changes in cell physiology.

Sustaining proliferative signaling

When proliferative signaling is sustained, growth is stimulated constantly:

1. A growth factor binds to a growth factor receptor
2. The growth factor receptor activates molecules, for instance Ras
3. Active Ras phosphorylates kinases
4. Kinases cause cell cycle progression
5. Cell growth

The phenotype is dominant → only 1 allele needs to be mutated. Some tumor cells:

• Secrete their own growth factors
  • Become independent of growth factors from the outside
• Modify their cell surface receptors
  • The receptors are constantly activated → don’t need growth factors anymore
• Mutate their intracellular signal molecules
• Mutate transcription factors
• Mutate components of the cell cycle network

Evading growth suppressors

Normal body cells are in equilibrium with growth suppressors and growth promotors. There are several checkpoints for controlling this:

• R-point
• DNA-integrity checkpoint
• Wnt signaling

R-point:

The R-point is the restriction point. Here, the Rb protein plays an important part → phosphorylation of Rb is necessary to release the restriction point. The Rb pathway is mutated in virtually all types of tumors. The Rb pathway can be inhibited by itself or by other means such as:

• Loss-of-function mutations of growth inhibitors
  • TGF-b
  • INK4a
• Gain-of-function mutations of growth factors
  • Cyclin D
  • CDK4

Both mutations cause Rb to become hypophosphorylated.

DNA-integrity checkpoints:

DNA-integrity checkpoints monitor 3 things:

• Whether the DNA is not too damaged
• Whether there are not mutations
• Whether the chromosome is properly attached to the spindle

A key player in this is p53, which is also called the guardian of the genome. It can be activated by:

• DNA-damage
• Hyperproliferative signals
• Telomere shortening
• Hypoxia

Activation of p53 leads to:

• Cell cycle arrest
• Senescence → not being able to replicate anymore
• Apoptosis

In more than 50% of all human tumors, p53 is mutated. p53 protective pathways are affected in more than 90% of all tumors. The following happens:

1. Loss of p53
2. Loss of DNA-integrity checkpoints → reduced apoptosis and senescence
3. Proliferation of cells with DNA damage
4. Mutations and chromosomal aberrations → genomic instability

The Li Fraumeni syndrome is a hereditary mutation. It is a heterozygous mutation of p53 which leads to multiple primary tumors at young age. It is inherited dominantly. During development, in many tissues the second allele is lost → everyone gets cancer. It is recessive on cell level.

Wnt signaling:

In case of Wnt signaling, b-catenin is degraded by antigen presenting cells (APC):

1. Wnt stimulation
2. APC releases b-catenin
3. b-catenin stabilizes and drives

HC5: Hereditary aspects of cancer

Risk of developing cancer

The chance of getting cancer increases with age:

• 8% by the age of 65
• 25% by the age of 80
• 32% by the age of 100

If someone has a sibling with breast cancer and is 40-50 years old, the chance of developing breast cancer is 2x as high as usual. In case of colon cancer, the cancer is 3x as high. Among monozygotic twins, the risk is higher → genetic factors play an important role in the risk of developing cancer.

Breast cancer

Genetic factors:

There are 14.000 new diagnoses of breast cancer per year in the Netherlands, of which 3.500 die every year. 13% have a first degree relative.

Genetic factors play a role in whether or not someone develops breast cancer:

• High/medium penetrant genes
• Low penetrant genes/loci/SNPs

There are 200 SNPs (single nucleotide polymorphisms) that increase the risk of breast cancer → per SNP, the increase is 0,1%, but multiple SNPs can be present at the same time.

Genetic counseling:

Genetic counseling is useful to:

• Recognize patients and their families with inheritable cancer
• Improve morbidity and mortality by early recognition and treatment
• Psychosocial guidance and advice

Cancer syndromes:

Many cancer-causing genes have been discovered. Examples are:

• RB gene
• APC gene
• Mismatch repair genes
• BRCA1 and BRCA2
• CDKN2A
• MUTYH
• BAP1

Criteria for genetic testing:

1 of the following situations has to be present to test for genetic breast cancer (BRC):

• BRC occurs at <40 years
• Bilateral BRC or multiple tumors in 1 breast with 1 tumor <50 years
• First grade male with BRC
• BRC <50 years and prostate cancer <60 years in the same branch of family
• BRC <50 years and 1 or more first degree with BRC <50 years
• BRC and 2 or more first and/or second degrees with BRC, of which at least 1 <50 years
• Ovarian cancer irrespective of age
  • Have a 10% risk of a germline BRCA1 or BRCA2 mutation

BRCA1 and BRCA2:

BRCA1 and BRCA2 mutations are associated with inheritable breast cancer. They are DNA-repair genes. Chances of developing cancer if 1 of these mutated genes is present are high:

• Breast cancer
  • BRCA1: 60-80%
  • BRCA2: 60-80%
• Ovarian cancer
  • BRCA1: 30-60%
  • BRCA2: 5-20%

BRCA mutation carriers undergo different types of surveillance, starting at early age:

• Breast surveillance
  • 25-60 years: yearly MRI
  • 25-60 years: clinical breast examination
  • 30-60 years: yearly mammography
  • 60-75 years: population screening
• Ovaries
  • 35-40 years: prophylactic adnex extirpation
    • Ovaries and fallopian tubes are removed

For males with a BRCA2 gen, the risk of developing prostate cancer is 2-4x as high.

They undergo a blood test for PSA at the GP, every 2 years starting at the age of 45.

Li Fraumeni syndrome:

In case of Li Fraumeni syndrome (LFS), there is a mutation of the TP53 gen. LFS is characterized

HC7: Clinical relevance of genetic repair mechanisms

Introduction

BRCA and Lynch syndrome occur very frequently in the population. Both syndromes are caused by DNA repair genes.

Even though germline mutations are equally distributed through the genome, hereditary cancer cases associate with syndromes caused by mutations in DNA repair genes. On the other hand, cancers related to inherited APC-mutations are also relatively frequent.

Damage and clinical prognosis

There is an association between DNA repair defects and clinical prognosis. Lung cancers have much more mutations than breast cancers. However, if there are more mutations, a tumor doesn’t necessarily have a worse prognosis. There are several reasons for this:

• There can be too much DNA damage in cancer cells → essential cancer genes are hit → the cell dies due to excessive damage
• Point-mutations are not the only alterations that occur in a cancer cell → tumors with very few mutations may have very abundant chromosomal mutations

Colorectal cancer genetics and prognosis:

Colorectal cancer can develop in several different ways:

• A minority develops under very specific defects in the DNA repair machine
• A small proportion (less than 5%) develops under deficiencies in the proofreading domain of polymerase chains → accumulate to a lot of mutations in their coding gene
• 15-20% develops under the context of mismatch repair deficiency
• A large majority has a low number of mutations in their genome but a large chromosomal instability

Even though MMR-deficient (mismatch repair deficient) or POLE-mutated (polymerase mutated) tumors have more mutations than MMR-proficient tumors, they have a better prognosis up to stage 3. In stage 4, the prognosis is equally bad for all tumors. This means that more mutations actually result in a better prognosis. This can be explained by:

• There being too much damage → affects the tumor cells
• Chromosomal instability leading to a worse prognosis than mutations

MMR-deficient and POLE-mutated tumors have a lot of lymphatic infiltrates in their tumors.

Endometrial cancer and prognosis:

In endometrial cancer, MMR-deficient and POLE-mutated tumors are also present. Here, POLE-mutated patients do better than patients who are MSS (microsatellite stable) or MSI (microsatellite instable). MSI-patients are MMR-deficient and MSS-patients are MMR-proficient. POLE-mutated and MSI patients have more lymphocyte infiltrates, which mainly consist of CD8 T-cells, than MSS-patients.

BRCA status and breast cancer prognosis:

Approximately 10% of all breast cancers carry defects in the BRCA genes, of which half are due to germline mutations. There has been a long debate about whether there is a relation between prognosis in breast cancer and the status of BRCA mutations. Recently, a big study has shown that there is no difference between BRCA-positive and BRCA-negative patients.

Arguments supporting an association between BRCA1 mutations and worse prognosis are:

• 70-90% of BRCA1 related tumors are triple-negative
  • Don’t express estrogen-receptors or HER2-receptors

Arguments against an association between BRCA1 mutations and a worse prognosis are:

• BRCA-related cancers are detected at earlier stages
• If looking at triple-negative tumors, BRCA mutations are associated with

HC9: Nomenclature and grading of cancer

Nomenclature

Mesenchymal tumors:

There are many kinds of mesenchymal tumors:

• Fat: lipo-
• Bone: osteo-
• Fibrous: fibr-
• Cartilage: chondro-
• Blood vessels: hemangio-
• Muscle: leiomyo-/rhabdomyo-

These tumors can be benign of malignant:

• Benign: -oma
  • Cells are nicely organized
• Malignant: -sarcoma
  • Sarcoma means flesh → malignant tumors are fleshy
  • Cells aren’t organized

Epithelial tumors:

Epithelium is the coverage and lining of the body. Tumors can be:

• Glandular or ductal
• Squamous and stratified

Epithelial tumors can be benign of malignant:

• Benign
  • Adenoma
    • Cystadenoma: a cyst originating from a duct
  • Papilloma
• Malignant
  • Carcinoma
    • Adenocarcinoma: carcinoma in the colon
    • Papillary carcinoma

Melanocytes:

Melanocytes can be benign or malignant:

• Benign
  • Nevus: a mole
• Malignant
  • Melanoma
    • In this case, “-oma” stands for a malignant disease

Testicular epithelium:

Tumors in testicular epithelium are always malignant → seminomas.

Totipotent cells can form all kinds of tissues. They are also located in the testis and ovaries. Tumors are called teratomas:

• Benign
  • Mature teratoma
• Malignant
  • Immature teratoma/terato-carcinoma

In women, teratomas are often benign. They can contain all types of tissues, even complete teeth.

Salivary glands:

Salivary glands have many different features. Tumors in salivary glands can vary greatly. They can be benign or malignant:

• Benign
  • Pleiomorphic adenoma
    • Has a high recurrence
  • Many others
• Malignant
  • Mucoepidermoid carcinoma
  • (Basal) adenocarcinoma
  • Acinic cell carcinoma

Grading

Grading defines the impact of grade and stage in different malignancies and can explain the differences between these features. A grade can be used as a biomarker, which is:

• Prognostic: for example of the chances of metastasis
• Predictive: for example of the chances of response to certain types of therapy

For all tumor types, there are different grading systems.

Grading of benign tumors:

There are 4 grades for benign tumors:

• Grade 1: well differentiated → low grade
• Grade 2: moderately differentiated → intermediate grade
• Grade 3: poorly differentiated → high grade
• Grade 4: undifferentiated → high grade

Bloom and Richardson:

Bloom and Richardson made grades which can be used to predict the survival rate, for instance for breast cancer:

• Grade 1 → 75% after 5 years
• Grade 2 → 53% after 5 years
• Grade 3 → 31% after 5 years

Fuhrman grading system:

The Fuhrman grading system can be used in renal cell carcinomas. These cells have clear cytoplasms. The bigger the nucleoli (dots in the nuclei), the higher the grade. There are 4 grades in total.

Gleason scoring system:

The Gleason scoring system can be used in case of prostate cancer. It is based on the amount of nicely formed ducts:

• Grade 1: nearly normal cells
• Grade 2: some abnormal cells, loosely packed
• Grade 3: many abnormal cells
• Grade

HC11: General principles: molecular diagnostics

Molecular diagnostics

Molecular diagnostics is a subspecialty of pathology that utilizes molecular biology techniques to:

• Detect normal and disease states
• Make a prognosis
• Identify druggable targets

Molecular biology techniques utilize DNA, RNA and enzymes that interact with nucleic acids to understand biology at a molecular level:

• KRAS mutations
• EGFR deletion
• EML4-ALK or ROS translocations in NSCLC
• Et cetera

Therapeutic targets

(Proto)oncogenes have several therapeutic targets based on the hallmarks of cancer:

• Growth factor
• Growth factor receptor
• Intracellular signal transduction pathway
• Cell cycle regulators
• Transcription factors
• Anti-apoptotic factors

Abnormal activation of these targets leads to cancer. This activation can be caused by:

• Dominant mutations at cellular level
• Single mutations → 1 allele is sufficient

Aneuploidy:

In a tumor cell, chromosomes become heavily dysregulated. This can even lead to entire chromosomes changing. When the chromosome number changes, it is called aneuploidy.

Type of mutations

Activating mutations can be divided into 3 categories:

• Translocation
• Point mutations
  • Missense mutations
    • An amino-acid is changed into another amino-acid
  • Nonsense mutations
    • A stop codon is introduced instead of an amino acid → truncated protein
  • Silent mutations
    • Even though a base pair is changed, it still codes for the same amino acid
  • Indel/frameshift mutations
    • A base is deleted and the next nucleotide is taken for translation
• Amplification
  • A normal gene becomes amplified and has multiple copies

Use:

Molecular diagnostics are useful to diagnose several things:

• Tumor phenotype
• Malignancy
• Origin of the tumor
• Heredity

Therapy:

Therapy can consist of:

• Personalized medicine
• Treatment of cancer
• Drugs targeting specific mutations/pathways

Material available for testing:

The vast majority of tissue is frozen and put into paraffine slides. This can be used for histological images → the location of tumor cells can be identified.  

Tumor heterogeneity

A tumor consists of:

• Parenchym
  • Real cancer cells/neoplastic cells
    • Have the actual mutations
    • Surrounded by macrophages, fibroblasts, etc.
• Stromal cells
  • Connective tissue and vessels
  • Different type of normal cells

Lots of lymphocytes are visible around tumor cells.

Detection of mutations

A tumor is a mix of neoplastic cells and supporting “normal” cells. The tumor cell percentage is the estimated % of tumor cells. Tumor cells are genetically instable and have a large variety. They can be distinguished as follows:

• Normal cell
  • 2 wild type alleles
• Tumor cell
  • 1 wild type allele
  • 1 mutation

Testing

There are 87 FDA approved drugs. For each drug, there is a molecular target which needs to be tested in the lab for 45 different disease indications. Every drug acts upon 1 hallmark of cancer.

BRAF:

A BRAF melanoma is caused by a BRAF mutation of the BRAF inhibitor. The following is found:

• Biopsy: neoplastic tissue with <1% tumor cells
• Microdissection: approximately 10% neoplastic

HC13: Heterogeneity in cancer

Types of heterogeneity

In cancer, there are 3 types of heterogeneity:

• Inter-patient: between patients
• Inter-tumor: between tumors
• Intra-tumor: within the tumor

Examples are:

• Well/moderately differentiated versus poorly differentiated
• Tumors with a strong T-cell infiltration versus with a poor T-cell infiltration
• Cancer cells are different when they are at the core than when they are at the invasive margin
  • Because the environment is different
    • For example the amount of immune cells or the access to oxygen
• The more clones there are in a tumor, the more likely the chance that 1 is able to metastasize

Heterogeneity forms a major issue for the treatment of cancer.

Intra-tumor and inter-tumor heterogeneity

Intra-tumor heterogeneity:

There are 2 origins of intra-tumor heterogeneity:

• Every time a tumor cell divides, it is an opportunity for a new clone to arise
• DNA repair deficient tumors are likely to be more clonal heterogenous

The fact that a new clone evolves, doesn’t mean it will survive:

• It has to compete with other clones
  • Competition for nutrients/oxygen
  • Proliferation
• It has to evade the immune system

Inter-tumor heterogeneity:

Inter-tumor heterogeneity is a morphological heterogeneity between tumors → one tumor is well/moderately differentiated while the other is poorly differentiated.

Heterogeneity becomes clear when the protein expression is observed. For instance, 1 tumor can have high levels of PD-L1 expression while the other doesn’t. Tumor cells which are negative for PD-L1 respond negative to the treatment.

Not only the tumor cells present in tumors can differ, but also other cells, for instance immune cells.

Origins of cancer cell heterogeneity:

Heterogenic tumors arise as follows:

1. Epigenetic changes occur during cell division
2. A clone that is able to evade the immune system survives and keeps dividing
3. The clone keeps getting better at evading the immune system

Of all the clones, 1 clone can remain resistant to therapy.

Clonal cooperation:

Clonal cooperation refers to characteristics of cancer provided by distinct cancer cell clones. Different clones, which all are present in the same tumor, have different functions:

• Clone A: tumor proliferation
• Clone B: angiogenesis
• Clone C: invasion

Clonal competition:

Aside clonal cooperation, there is competition between cancer cells. Cells compete for nutrients, space and oxygen. Many clones that are generated during tumor progression do not survive. This competition is driven by selective pressure, which is driven by either external factors or intrinsic features of cancer cells:

• Immune recognition
• Competition of nutrients/oxygen
• Inter-clonal competition

Inter-patient heterogeneity

Different genes are mutated in different cancers. Only TP53 mutations occur in nearly every type of cancer. The large majority of mutated genes differs considerably from one tumor to the other tumor type. They play different roles in healthy tissues. This inter-patient heterogeneity has consequences for the treatment.

An example is HER2, which is mainly expressed in breast and urothelial cancer. These cancer types can be treated with trastuzumab.

Different mutation profiles:

Inter-patient heterogeneity can

HC15: Framework oncology and staging

Tumor grading

A tumor grade reflects intrinsic biological behavior of tumors. In general, a low grade is less aggressive. It is necessary to grade a tumor for treatment and prognosis:

• Lower grade → better prognosis
• Higher grade → worse prognosis

Tumors are graded based on the microscopic appearance of cancer cells. Dependent on the tumor, there are 2-4 degrees of severity. An example is:

• GX: grade cannot be assessed
  • Undetermined grade
• G1: Well-differentiated
  • Low grade
• G2: moderately differentiated
  • Intermediate grade
• G3: poorly differentiated
  • High grade
• G4: undifferentiated
  • High grade

Breast cancer grades:

The amount of grades differs per tumor type. In case of breast cancer, there are only 3 grades. These grades can be linked to the expected 5 year survival rates:

• Total: 91-95%
• Grade I: 75%
• Grade II: 53%
• Grade III: 31%

Staging systems

Tumor staging is used to determine the extent of disease spread in patients:

• Solid tumors
  • Local spread
  • Through lymphatics (lymphogenic)
  • Distant (hematogenic)
• Haemotological malignancies
  • Tells something about the disease extent and influence on normal bone marrow function

Cancer staging is useful for:

• Planning treatment
• Estimating prognosis
• Identifying clinical trials/studies
• Making a comparison between institutes
• Communication
  • Staging is a universal language

There is no unique staging system. However, most staging systems do consist of several common elements:

• Location of the primary tumor
• Tumor size and number of tumors
• Lymph node involvement
  • Spread of cancer into the lymph nodes
• Cell type and tumor grade
  • How closely the cancer cells resemble normal tissue
• Presence or absence of distant metastasis

In general, the higher the stage, the worse the prognosis.

TNM staging principles:

The TNM staging principles is the most used staging system for solid tumors:

• T-status: extent of the tumor
  • T1-T4
    • The larger the tumor, the higher the T
  • Size of the tumor and locally organ involvement
• N-status: regional lymph node involvement → absence or presence of metastasis
  • N0: no lymph nodes are involved
  • N1: 1 or 2 lymph nodes are involved
  • N2: infra- or subclavicular lymph nodes are involved
• M-status: distant metastasis
  • M0: absent
  • M1: present

The rules per tumor differ → prefix modifiers can be useful:

• cTNM → c = clinical
  • Before treatment
  • Mostly based on imaging or physical examination
• pTNM → p = histopathological
  • After surgery
• ypTNM → yp = pathological after pretreatment
  • After chemotherapy, but before surgery
  • There is a large difference between the c and p stage
• TNMx → x = not classified
  • Nothing is known about the tumor

For instance, a PA report states the following:

• Ductal adenocarcinoma
• Grade 3
• Diameter: 1,2 cm
• Free excision

HC18: Biomarkers for early detection of cancer

Biomarkers

Cancer can be diagnosed based on:

• Physical examination
• Blood tests
• CT-scans
• Biopsies to obtain tumor tissue

A biomarker refers to a measurable indicator of some biological state or condition. Humans shed particles into the bloodstream or environment as evidence of their presence in a particular location:

• Small parts of cells
  • Microparticles
  • Exosomes
• Proteins
• Unique chemicals
• DNA and RNA

Biomarkers are measured and evaluated to examine:

• Normal biological processes
• Pathogenic processes
• Pharmacologic responses to a treatment
  • Therapeutic intervention

An ideal tumor marker for diagnosis:

• Should have great sensitivity, specificity and accuracy in reflecting the total disease burden
• Should be prognostic of the outcome
• Should be predictive of tumor recurrence
• Should be predictive of effectiveness of anti-cancer treatments

Samples for biomarker detection are:

• Blood, urine or other body fluids
• Tissues

Tumor cells and products circulate in blood and are easily detectable. However, tissue forms the issue. There is a limited access to tissue:

• Archival tissue is not obtained at the time of clinical decision
• Accessible metastatic tissue may not be representative

Circulating tumor cells

Because tissue access is limited, there is an increased interest in circulating tumor cells (CTCs). CTCs shed by primary and metastatic tumors can be used as “liquid biopsies”, providing real-time information about the patient’s current disease state. Molecular profiling in CTCs can be used for patient selection to stratify for targeted therapy or serve as well-defined treatment targets.

Clinical applications

Potential clinical applications of CTCs are:

• Blood testing using CTCs may aid in cancer diagnosis
• May serve as prognostic and predictive biomarkers
  • Changes in CTC counts could indicate sensitivity or resistance to anti-cancer therapy monitoring
• CTC enumeration may estimate tumor burden and tumor invasiveness
• Investigational platform to eludicate tumor biology
  • Whole genome/transcriptome sequencing

Microparticles:

CTCs are not whole tumor cells → they are circulating biomarkers. CTCs are very rare in the blood of cancer patients, ranging from 1 to over 1000 in 10 ml blood samples, which usually contain 50-100 billion red and white blood cells.

Microparticles from a tumor are released into the bloodstream. Microparticles are:

• CTCs
• Genomic DNA
• miRNA
• Microvesicles (MV)
• Microparticles (MP)

Clinical uses

Clinical uses of biomarkers are:

• Screening
• Making a diagnosis
• Marker of prognosis
• Monitoring of treatment efficacy
• Detection of recurrence of the disease

Screening:

A biomarker for screening must be:

• Highly specific (for a single type of cancer) → minimize false positive and negative
• Able to clearly reflect the early stage of disease
• Easily detectable without complicated medical procedures
• Cost effective

Monitoring of treatment efficacy:

A biomarker for monitoring treatment efficacy must:

• Correlate with tumor size/volume
  • The actual number of tumor cells in the body → tumor load
• Short in half-life circulation
• Be highly specific and sensitive for a single type of cancer

The ideal tumormarker

HC20: Radiation oncology

Radiation oncology

Radiation oncology is a separate discipline in oncology where ionizing radiation is used to treat cancer. It is not the same as radiology or nuclear medicine:

• Radiation oncology uses as much radiation as possible, radiology uses as little radiation as possible
• Nuclear medicine is done with the use of soluble radionuclides, radiation oncology uses linear accelerators and fixed sources

50% of cancer patients are irradiated. Radiotherapy can be a form of curative or palliative treatment. Together with other treatments, it increases the numbers of long survivors.

Ionizing radiation

There are several types of ionizing radiation:

• α-radiation
  • Low penetrance
    • Can be stopped by a piece of paper → not often used
  • Sometimes used with cell cultures to measure
• β-radiation
  • Often used
• γ-radiation
  • Most used
• H⁺-radiation (protons)
  • Sometimes used
• Electrons
  • Sometimes used
  • Low penetrance
    • Ideal for superficial tumors (skin cancers) → doesn’t affect deeper laying tissues

External beam radiotherapy:

External beam radiotherapy uses the photons of γ-rays. A linear accelerator can be turned on or off. This is a form of local treatment → there only is radioactivity when the linear accelerator is on. Afterwards, patients won’t be radioactive anymore.

Mechanism of action

Radiation oncology has the following mechanism of action:

1. Radiation causes DNA-strand breaks
   • This can happen due to direct photon-DNA interaction, but mostly due to indirect interaction: photons activate oxygen radicals → cause DNA damage
   • Double strand breaks, single strand breaks and other kinds of DNA damage are being caused
2. Normal cells recognize and repair most of the damage
3. Cancer cells have less regenerative capacity and die at cell division

The aim is to kill the tumor. The side effects of the therapy are determined by the slow down of cell division in surrounding healthy tissues. This causes both acute and late cell side effects.

Dose severity

A higher dose does not always give more tumor control → the likelihood of tumor control isn’t a linear line but is S-shaped. The higher the dose, the higher the toxicity to both tumor and normal cells. The aim of radiotherapy is to kill the tumor without normal cell damage.

Therapeutic window:

The therapeutic window or safety window refers to a range of doses which optimize between efficacy and toxicity, achieving the greatest therapeutic benefit without resulting in unacceptable side-effects or toxicity. It is the space between the tumor control curve and toxicity curve.

There are ways to increase the therapeutic window:

• Make the tumor more sensitive to radiotherapy
  • The tumor responds much faster than the normal healthy cells
  • The therapeutic window becomes bigger → more tumor control with the same amount of radiation
• Make the healthy tissue more resistant to radiation
  • A bigger therapeutic index

Increasing the therapeutic window enhances the sensitivity.

Side effects

Radiation therapy has early,

HC22: Chemoradiation

Enhancement of sensitivity

The therapeutic window can be increased by:

• Fractionation of the total dose
• Decreasing the toxicity → lower the dose to normal tissue
• Increasing the tumor control → enhancing the sensitivity

The sensitivity can be enhanced via:

• Chemoradiotherapy
  • Radiotherapy + chemotherapy
• Biological agents
  • Cetuximab in head and neck cancer
• Oxygen to tumor tissue
  • Increases the oxygen which reaches the tumor → high response rate
  • Doesn’t really work
• Hyperthermia
  • Exposing tumor tissue to high temperatures
  • In patients with cervical cancer and relapses of breast cancer

Concomitant and sequential

Chemoradiation is a combination between radiotherapy and chemotherapy. This can be:

• Concomitant: in the same time period
• Sequential: behind each other

Mechanism of action

If chemoradiation is used, DNA damage to tumor cells is more often fatal than with irradiation alone. Normal cells have a greater variety of escape mechanisms and repair the damage, which causes them to be more able to repair DNA than tumor cells → have a higher tolerance to the therapy. There are different modes of action for radiation and chemotherapy. Chemotherapy is used as a radiosensitizer which enhances the effect of radiotherapy.

Synergy:

Chemo- and radiotherapy can be used to create synergy:

1. Cisplatin adducts to DNA → causes single strand breaks
2. Radiotherapy will create more breaks which are more difficult to repair
3. Post-irradiation repair is inhibited

Chemotherapy inhibits nucleotide metabolism, while radiation is effective in a different phase of the cell. Chemotherapy treats hypoxic cells, which are less radiosensitive for radiotherapy. Chemoradiation is seen as local treatment to increase local control and thereby cure.

Uses of chemoradiation

Chemoradiation is only used in curative treatment:

• As a primary treatment modality
  • Organ saving treatment
  • Curative resection is not possible
• As adjuvant treatment
  • Increases local control after resection
• As neoadjuvant treatment
  • Increases local control after resection

Primary treatment:

As primary treatment, chemoradiation can be used as an organ saving treatment. This can be useful when curative resection causes a large loss of function and therefore isn’t possible or when the patient isn’t operative anymore. Examples are:

• Anal cancer
  • When surgery is done, the patient will most likely lose his anus and need a stoma
• Head and neck tumors
  • E.g. in case of larynx cancer: if a patient loses their voice, the quality of life decreases significantly
• Cervical carcinoma
• Vulvar cancer
• Esophageal cancer
  • Not operable
• Stage III lung cancer

Adjuvant treatment:

Chemoradiation as adjuvant treatment increases the local control after resection, for example in case of stomach cancer. It can prevent recurrence.

Neoadjuvant treatment:

Chemoradiation as neoadjuvant treatment:

• Increases the local control after resection
• Downstages resectable cancer

Examples are operable esophageal cancer and advanced stage rectal cancer.

Side effects

Chemoradiation has much more acute side effects in contrast to surgery:

• Acute side

HC24: Diagnostics in hematology

Case

A 40-year-old man visits the GP with complaints of fatigue and pallor. The GP thinks the most likely clinical problem is anemia.

Automatic hematology analyzer

The GP requests a complete blood count (CBC) → a differential count of Hb, leukocytes, and thrombocytes. Blood goes through an automatic hematology analyzer, a laser which gets scattered when it encounters a blood cell:

• Forward scatter/low angle: shows the volume of the cell
• Side scatters/high angle: shows the complexity of the cell (granules, MPC, etc)

The machine is able to identify the cells based on size and complexity. The results are shown in a diagram, which makes it possible to determine how many and what kind of cells there are.

Results

Results of the CBC show:

• Hb: 4,3 mmol/L → too low
• Leukocytes: 40 x 10⁹/L → too high
• Platelets: 70 x 10⁹/L → too low

Acute leukemia is suspected. In leukemia, there are too many leukocytes compared to other cells. The diversity also is less.

Microscopic leukocyte differentiation

Microscopic leukocyte differentiation consists of a blood smear stained with May Grunwald Giemsa. In case of acute leukemia, differentiation of leukocytes is lost → many strange looking leukocytes which all look the same are present.

Levels of diagnostics

There are several levels of diagnostics which make it possible to examine different structures:

• Resections/biopsies: tissue
• Cytology: cells
• Molecular pathology: molecules

Bone marrow examination

The bone marrow is the principle site of hematopoiesis. Examination of the bone marrow provides additional information for diagnostic clues seen in the peripheral blood:

• Increased or decreased leukocytes
• Increased or decreased Hb
• Increased or decreased platelets
• Presence of abnormal or immature cells

Diagnostic tests:

Several diagnostic tests on the bone marrow tissue can be done:

• Standard histology: on tissue level
  • H&E: hematoxylin (nucleus) and eosin (cytoplasm)
  • Histochemical stainings (PAS, alcian blue, etc.)
• Immunohistochemistry: on tissue/cell level
• Molecular assays: on molecular level

Bone marrow aspirates are liquid components of bone marrow. On bone marrow aspirates, tests can be done as well:

• Hematomorphology
  • Distinguishes cell types based on morphological criteria
    • Blasts
    • Mature and immature cells
      • Myeloid
      • Erythroid
      • Megakaryocytic
    • Lymphocytes
    • Plasma cells
    • Macrophages
    • Mast cells
  • Count: 2 x 500 cells
  • Sensitivity: 1%
    • 1 abnormal cell in background of 100 normal cells
  • Expertise, experience and pattern recognition is necessary
  • Acute leukemia: there is way less diversity → everything looks the same
• Immunophenotyping (cytology)
  • Distinguishes cell types based on presence or absence of proteins or antigens
    • Blasts
    • Mature and immature cells
      • Myeloid
      • Erythroid
      • Megakaryocytic
    • Lymphocytes
    • Plasma cells
    • Macrophages
    • Mast cells
  • Count: 100.000-1.000.000 cells
  • Sensitivity: 0,0001-0,001%
  • Expertise, experience and pattern recognition is necessary
  • Antigens called clusters of differentiation markers (CD markers) are used to identify cells → specific combinations of CD markers on the cell surface are used to identify different cells
    • CD markers are attached to fluorescent

HC26: Malignant lymphomas

Principles of lymphoid differentiation

There are 2 types of malignant lymphomas:

• T-cell lymphoma/NK-cell lymphoma
• B-cell lymphoma

Pluripotent hematopoietic stem cells (HSC) differentiate into:

• Common lymphoid precursor cells (CLP) → differentiate into:
  • T-precursor cells → naïve T-cells → T-memory cells
  • B-precursor cells → B-memory cells or plasma cells
  • NK-cells
• Common myeloid precursor cells (CMP)

By morphology alone, CLPs cannot be distinguished from CMPs → additional tests are necessary. A plasma cell is recognizable thanks to its big Golgi system. This is the only type of cell which can be distinguished by cell morphology alone. The difference between T-cells and B-cells can be made by looking at cell type specific antigens which are present on the cell:

• T-cells: CD3
• B-cells: CD19, CD20, CD79
• NK-cells: CD16/56

Neoplasia:

The primary purpose of lymphocytes is to recognize an antigen receptor. Precursor cells, like blast cells, do not have antigen receptor (AgR) expression yet → they are AgR negative. Both AgR positive and negative cells can cause neoplasia:

• Precursor neoplasia: caused by AgR negative cells
  • Have a blast-like morphology
• Mature neoplasia: caused by mature (AgR positive) cells
  • Have a lymphocyte/plasma cell morphology

If mature cell receptors are destroyed, the cell goes into programmed apoptosis. This doesn’t happen in case of plasma cells, because they don’t have a receptor.

Physiological B-cell differentiation

There aren’t enough genes to code for the different type of receptors present in the adaptive immune system. This is solved by VDJ (IgH) and VJ (IgL) recombination:

1. A B-cell precursor starts to express a receptor to become a B-cell
2. The B-cell precursor starts to cut out parts of DNA to form a functional gene → multiple V and J sequences are put together
   • 18 different combinations can be made
3. More differentiation is possible by nibbling away some nucleotides on the open ends of DNA and adding random nucleotides
   • This happens by chance
4. A completely new functional gene with a completely unique receptor is made

Mechanism of B-cell lymphomas

Lymphomas are frequently caused by activation of proto-oncogenes in the course of antigen receptor formation:

• Chromosomal translocations
  • VDJ recombination
  • Class switch recombination
• Point mutations
  • Somatic hypermutation

This results in resistance to apoptosis and activation of signaling cascades.

B-cell maturation is a dangerous process because it contains double strand DNA breaks → risk of getting mutations. For instance, this can happen when VDJ-recombination activates a proto-oncogene:

1. During recombination an oncogene is translocated to an immunoglobulin heavy chain locus on the genome
2. The oncogene becomes irreversibly active
3. The B-cells containing oncogenes mature
4. In a lymph node, the mutated B-cell is exposed to an antigen for which its receptor is sensitive
5. The germinal center response takes place → the mutated B-cell develops from centroblast to centrocyte
   • Somatic hypermutation and class switch recombination takes place
6. The mutated B-cells have

HC29: HLA & minor histocompatibility antigens

Allogeneic SCT

There are 2 ways of doing an allogeneic SCT:

• Allogeneic SCT with T-cells present in the cell graft: transplantation of an immune system of a healthy donor with the aim to induce a strong T-cell response against the cancer cells of the patient
  • Mature donor T-cells in the stem cell transplant mediate GVL and GVHD
  • Systemic immunosuppression is required to suppress GVHD
  • Systemic immunosuppression is gradually decreased
• Allogeneic SCT without T-cells present in the cell graft
  • No systemic immunosuppression is required
  • Consists of 2 steps
    • T-cell depletion → no systemic immunosuppression is required
    • DLI is necessary: 3-6 months after allogeneic SCT to induce GVL
      • The patient is in better condition
      • Professional antigen-presenting cells of donor origin
      • Less pathogens
      • Less inflammatory cytokines
  • The T-cell response is weaker → better balance between GVL and GVHD
    • These patients do better as a group → lesser GVHD reaction
      • The body has time to heal damaged tissue from chemotherapy
      • Recipient dendritic cells can be replaced by donor dendritic cells

T-cells in infections

HLA:

HLA molecules are located on chromosome 6. Per chromosome, there are 6 genes which play a role in HLA. Because humans have 2 chromosomes, there are 12 genes in total which play a role. There are 2 types of HLA molecules:

• HLA class I: present intracellular antigens
  • HLA-A
  • HLA-B
  • HLA-C
• HLA class II: present endocytosed antigens
  • HLA-DR
  • HLA-DQ
  • HLA-DP

The HLA groups are located in the peptide binding groove. Only dendritic cells, macrophages and B-cells are capable of HLA-II expression. HLA is highly polymorphic → there are many variants and every different allele has its own name.

Negative selection:

Due to negative selection in the thymus of T-cells which have high affinity for HLA self-complexes, there are no T-cells for processing peptides derived from cellular proteins, otherwise autoimmune reactions would be induced.

T-cells after allogeneic SCT:

Donor T-cells recognize foreign peptide-HLA complexes (allo-antigens). When selecting a matching donor, not all 12 but only 10 genes are taken into account → HLA-DP is usually not taken into account:

• When an unrelated donor (URD) is selected, there usually is a 10/10 match but an HLA-DP mismatch
• When a family donor (sibling donor) is selected, there usually is a 12/12 match with HLA-DP also matching

Therefore, after an unrelated allogeneic SCT, there can be T-cells present in the donor graft which are directed against peptides in mismatched HLA-DP → immune reaction. HLA molecules are major histocompatibility antigens. In case of shared HLA molecules, immune responses against minor histocompatibility antigens can occur.

Minor histocompatibility antigens

A minor histocompatibility antigen (MiHA) can be:

• A polymorphic peptide that differs in amino acid composition between patient and donor
• A polymorphic peptide that is presented on patient cells by HLA surface molecules
  • HLA is matched between patient and donor

HC31: Targeted therapy and hematological malignancies

Monoclonal antibodies

Tumor specific monoclonal antibodies:

CD20 is an IgG isotype expressed antigen on normal and malignant B-cells. It is used for treatment of many B-cell lymphomas → by targeting CD20 antigens, both malignant and normal B-cells are depleted.

Rituximab:

Chemotherapy combined with CHOP monoclonal antibodies leads to a significant increase of survival rates. Rituximab, a monoclonal antibody against CD20, leads to:

• Complement system activation
  • A MAC is formed
• NK-cell recruitment
  • Receptors bind to the humanized tail of rituximab
• Apoptosis
  • Macrophages are attracted

Mechanisms of action:

Monoclonal antibodies can have different mechanisms of action:

• Complement dependent cytotoxicity (CDC)
  1. The complement system is activated
  2. Hollow pipes are made in the cell surface
  3. Water enters the cell
  4. The cell explodes
• Antibody dependent cellular cytotoxicity (ADCC)
  1. A B-lymphocyte plasma cell secretes antibodies which bind to an antigen on the pathogen
  2. The pathogen is phagocytosed by macrophages, destroyed by NK-cells or lysed via the complement system
• Radio immunotherapy
  1. A radioactive particle such as yttrium is coupled to an IgG monoclonal antibody such as CD20 → can get close to the tumor cells
     • A form of local treatment
  2. Yttrium gives a small dose of β- or γ-radiation
  3. The tumor is directly targeted
• Antibody drug conjugate
  1. A drug is bound to a monoclonal antibody such as CD30 → an ADC-CD30 complex is created
     • CD30 is expressed in Hodgkin lymphomas and anaplastic large cell lymphomas
  2. The ADC-CD30 complex travels to the lysosome
  3. MMAE is released in the cell → disrupts the microtubule network
     • Physical cell division is prevented by binding on a microtubule during cell division
  4. The G2-/M-phase is arrested → the cell cycle stops → apoptosis

Bispecific antibodies

Bispecific antibodies are a combination of 2 different antibodies to target B-cells with CD8 T-cells. A bispecific T-cell engager (BiTE) causes proliferation of T-cells via the following mechanism:

1. 2 antibodies are coupled to create a bispecific monoclonal antibody with binatunumab → an antibody against CD3 antibody is coupled with an antibody against CD19
   • Anti-CD3 antibodies target T-cells
   • Anti-CD19 antibodies target B-cells
2. The T-cell becomes activated and starts its immune function → cytokines are made
   • A T-cell can kill more cells than 1 B-cell
3. The T-cell attacks malignant B-cells → perforins enter the B-cells and kill them

CAR T-cells

A T-cell has a T-cell receptor, which can recognize a peptide presented in an HLA molecule. To activate a T-cell receptor, additional costimulatory signals such as CD28 are necessary as well. If a T-cell receptor is activated, the T-cell proliferates and kills the target cell. A Chimeric antigen receptor (CAR) T-cell uses the intracellular part of a TCR and the extracellular part of a monoclonal antibody:

1. T-cells are extracted through a leukapheresis procedure
2. T-cells are expanded if necessary
   • Usually

HC34+35: Secondary hemostasis I&II

Secondary hemostasis

Blood flows as a fluid through the blood vessels to all the organ systems. Upon injury, blood vessel loss has to be prevented. This is done via blood coagulation. Under normal conditions, blood coagulation is a tightly regulated process:

• Rapid formation of a clot upon injury
• Limited to the site of injury

Secondary hemostasis is the conversion of soluble fibrinogen to insoluble fibrin by thrombin (IIa):

• Strengthens the platelet plug
• Induces adherence and activation of cells involved in vascular repair
  • Fibroblasts
  • Smooth muscle cells

Thrombin plays a key role in the conversion of fibrinogen to fibrin. It sometimes also is called coagulation factor IIa.

Fibrinogen

Fibrinogen is also known as factor I and is very abundant in the plasma → normal levels are 1,5-3 gram/L. It consists of 2 symmetrical half-molecules, each consisting of 3 different polypeptide chains:

• Aα: linked together in the E-domain → the central parts
• Bβ: in the D-domain → the outer parts
• γ: in the D-domain → the outer parts

Formation of fibrin:

Fibrinogen is formed into fibrin as follows:

1. Thrombin cleaves away fibrinopeptides A and B → fibrin monomers are formed
   • Fibrin monomers are very sticky
2. 2 fibrin monomers stick together → dimers are formed
   • This is done via H-bonds
3. Coagulation factor XIIIa crosslinks different dimers → polymers are formed
   • Coagulation FXIIIa = transglutaminase
     • The enzyme factor XIII is converted into FXIIIa by thrombin (IIa)
   • The crosslinks are formed between the D-domains of the fibrin
   • These are covalent bonds

Thrombin

Thrombin is generated via the coagulation cascade. The coagulation cascade is a sequence of proteolytic reactions, which take place in parts of the surface of activated proteins. In each step, a pro-enzyme is converted to an enzyme, which activates the next pro-enzyme. These are slow reactions which can be sped up by co-factors → a kind of catalysts.

The coagulation cascade consists of 2 pathways:

• Extrinsic pathway: activation upon contact with tissue factor (TF)
  • TF was previously named coagulation factor III
  • The initiator is located outside the plasma
• Intrinsic pathway: activation upon contact with surfaces foreign to our body
  • E.g. glass → contact activation
  • Everything is located inside the plasma

The common pathway consists of the coagulation factors which play a part in both intrinsic and extrinsic pathways:

• FX
• FV
• FII

Extrinsic pathway:

There are several very important factors in the extrinsic pathway:

1. Tissue factor, factor VII
2. Factor X, factor V
3. Factor II: prothrombin

Tissue factor (TF) is a transmembrane glycoprotein and a non-enzymatic co-factor of FVIIa. TF is produced by fibroblasts and smooth muscle cells → is expressed on a "hemostatic envelope” around blood vessels. TF normally isn’t present in the blood compartment, but during pathological conditions it can be expressed:

• Inflammation: expression on endothelial cells and monocytes
  • This is

HC37: Cancer, coagulation and thrombosis

The link between cancer and thrombosis

Cancer leads to coagulation, which leads to thrombosis. Coagulation itself also leads to cancer, and thrombosis may as well. The link between cancer and thrombosis was established almost 200 years ago by Jean-Baptiste Bouillaud and Armand Trousseau. Trousseau diagnosed himself with thrombosis and predicted he suffered of cancer. Months later, he indeed died of pancreatic cancer.

Dual problem:

Cancer and thrombosis are a dual clinical problem. Out of 7 million cancer-associated deaths, 1 million are attributed to thrombotic complications. Patients with cancer and thrombosis have an extremely bad prognosis. The second main cause of death in cancer patients is deep venous thrombosis/pulmonary embolism (VTE):

• 25% of all first VTE events are cancer-related
• The presence of malignancy results in a 7x increased risk of VTE
  • 0-3 months after cancer diagnosis: 54x increased risk
  • 3-12 months after cancer diagnosis: 14x increased risk
  • 12-36 months after cancer diagnosis: 4x increased risk
• 8% of all cancer patients experience VTE

The risk of developing VTE in case of cancer can be divided into 2 groups:

• Clinical risk factors
• Biological risk factors

Clinical risk factors

Stage:

Not all cancer types confer the same risk for VTE. Patients with pancreas, lung and/or brain cancer have the highest incidence rate of VTE. The more aggressive the tumor, the higher the risk. The risk of VTE reflects the stage of the disease → in remote cancers, the risk of VTE is highest.

Therapy:

Certain cancer treatments can increase the risk of VTE as well:

• Surgery: 2x increased risk of VTE in cancer patients
• Chemotherapy: the risk varies
• New antiangiogenic drugs
• Prolonged bed rest

Treatment of venous thrombosis in cancer patients usually consists of low molecular weight heparin (LMWH), which is more effective than vitamin K agonists. LMWH can be prescribed for 6 months. If VTE still is present after this, it is necessary to switch to vitamin K antagonists.

Biological risk factors

It is unknown which biological factors cause cancer-associated VTE. Possible causes are:

• Microparticles
• Neutrophil extracellular traps (NETs)
• Coagulation factors
• Localization of cancer cells to the blood (CTCs)
• Platelets/leukocytes
• Compression of blood vessels by the tumor

Microparticles:

Microparticles (MPs) are vesicles of 50 nM-1 μM shed from various cells. They are meant for intercellular communication and contain proteins and microRNA. TF+ microparticles (MP-TF) can be shed from:

• Platelets
• Leukocytes
• Endothelium
• Cancer cells

MP-TF predicts the chance of VTE → if more MP-TF+ is present, the cumulative incidence of VTE is higher:

• Healthy volunteers have low TF activity in plasma
• Cancer patients without VTE have low TF activity in plasma
• Cancer patients with VTE have high TF activity in plasma → microparticles play a role in VTE

A hypothesis is that once MP-TF is in the blood, it fuses with platelets, endothelial cells or the ECM. MPs can be measured with:

• Fluorescence-assisted cell sorting (FACS)
  • Cells flow through a machine

HC39: Thrombosis

Cause

Thrombosis is caused by a decrease in anticoagulant factors. Deep vein thrombosis (DVT) has the following symptoms:

• Red skin
• Warm
• Painful calf
• Swelling
• Shining skin
• Pain while walking

In 40% of cases with suspected DVT, the clinical diagnosis is correct. Ultrasounds of the vessels in the leg are primarily used to diagnose DVT. In normal situations, the vein compresses and becomes hard to see. In case of thrombosis, the vein doesn’t become compressed as much. DVT differs from arterial thrombosis because it occurs at sites of stasis.

Post thrombotic syndrome

Post thrombotic syndrome (PTS) is a persistent swollen and painful leg as a result of DVT. Symptoms are:

• Swollen leg
• Heavy feeling leg
• Pain
• Varicose veins
• Discoloration of the skin
• Ulceration

In post thrombotic syndrome, accumulation of thrombotic platelets causes a leaky valve. Current treatment to prevent the development of post-thrombotic syndrome consists of compression stockings → the venous pressure is increased.

Pulmonary embolism

A pulmonary embolism (PE) is an embolization of a venous thrombus into the pulmonary artery. Symptoms are:

• Breathlessness
• Pleural pain
• Hemoptysis
• Fever
• Shock

A very deadly form of pulmonary embolism is a saddle embolism → almost always results in death.

Imaging:

Diagnosis of pulmonary embolism requires objective imaging:

• CT-scans
  • Are primarily used
  • White contrast fluid is injected → gray areas show blood clots
• Chest X-rays
  • In acute phases of pulmonary embolisms
  • Rules out other diseases mimicking pulmonary embolism
  • After a few days, pulmonary infiltrates may become visible → infarcts
• Ventilation-perfusion scan: shows radioactivity in the lungs → if there is radioactivity, no blood is present
  • Perfusion: intravenous injection of radioactive technetium macro-aggregated albumin
    • Tc99m-MAA
  • Ventilation: inhalation of gaseous radionuclides in aerosol
    • Xenon-133
• Pulmonary angiography
  • Was used in the past
  • Contrast fluid is injected into the pulmonary artery with a catheter

Diagnostic strategies:

Diagnostic strategies for pulmonary embolism may consist of an algorithm, such as the clinical decision rule:

• Symptoms of DVT → 3
• Heart rate >100/min → 1,5
• Immobilization >3 days or operation in the past 4 weeks → 1,5
• DVT or PE in medical history → 1,5
• Hemoptysis → 1
• Malignancy → 1
• PE is more likely than any alternative diagnosis → 3

In case there are less than 4 points, a D-dimer test is done:

• D-dimers <500 ng/ml → no PE
• D-dimers >500 ng/ml → possibly PE

In case there are more than 4 points or D-dimers are >500 ng/ml, a CT or perfusion scan is made:

• Normal → no treatment
• PE → treatment
• Other diagnosis → treatment

Currently, a more simple way is used → the YEARS algorithm. If 3 YEARS items are present, a D-dimer test needs to be ordered:

• Clinical signs of DVT
• Haemoptysis
• If pulmonary embolism is the most likely diagnosis

This prevents a lot of unnecessary CT-scans.

Treatment

The primary aim